ABSTRACT
Post-transcriptional gene silencing (PTGS)-mediated gene silencing exploits the cellular mechanism whereintranscripts having sequence similarity to the double-stranded RNA (dsRNA) molecules present in the cell will besubjected to degradation. PTGS is closely related to natural processes such as RNA-mediated virus resistance andcross-protection in plants. Gene silencing and the cellular machinery for affecting this phenomenon might haveevolved as a natural protective measure against viral infection in plants. In PTGS, small interfering RNA (siRNA)molecules of 21–23 nucleotides length act as homology guides for triggering the systemic degradation of transcriptshomologous to the siRNA molecules. PTGS phenomenon, first discovered in transgenic petunia plants harbouringchalcone synthase gene and termed co-suppression, has been subsequently exploited to target specific gene transcripts for degradation leading to manifestation of desirable traits in crop plants. Targeted gene silencing has beenachieved either through the introduction of DNA constructs encoding dsRNA or antisense RNA or by deploying cosuppression constructs producing siRNAs against the transcript of interest. Understanding the mechanism of genesilencing has led to the development of several alternative strategies for inducing gene silencing in a precise andcontrolled way. This has paved the way for using PTGS as one ofthe chief functional genomicstools in plants and hashelped in unraveling the mechanism of many cellular processes and identifying the focal points in pathways, besides,opening new vistas in genetic engineering of plants for human benefits. PTGS has shown great potential in silencingthe deleterious genes efficiently so that value-added plant products could be obtained. Thus, PTGS has ushered in anew era in the genetic manipulation of plants for both applied and basic studies. In this review, we have outlined thebasics of RNAi-mediated gene silencing and summarized the work carried out at our institute using this approach, ascase studies. In particular, adopting RNAi-mediated gene silencing (a) as a method to restore fertility in transgenicmale sterile lines developed based on orfH522 gene from sunflower PET1-CMS source, (b) as a tool to suppress theproduction of toxic proteins, ricin and RCA, in castor, and (c) as an approach to induce bud necrosis virus resistancein sunflower has been discussed. Examples from other plant systems also have been mentioned to exemplify theconcept and utility of gene silencing in crop plants.
ABSTRACT
@# Objective: To explore the effect of interfering insulin-like growth factors-1 receptors (IGF-1R) by small interfering RNA (siRNA) on cell cycle and apoptosis of hypoxic hepatocellular carcinoma HepG2 cells. Methods: The hypoxic hepatocellular carcinoma model was established via cobalt chloride treatment. Three siRNAs targeting IGF1R gene and one negative control siRNA were designed and synthesized. They were transfected into hypoxic HepG2 cells, and 24 h later, the transfection efficiency was detected by fluorescent microscopy. The protein expression of IFG-1R was detected with Western blotting (WB) to screen the siRNA with highest transfection efficacy. The selected siRNA was used to transfect hypoxic HepG2 cells. The proliferation of hypoxic HepG2 cells was determined by MTT assay. Cell cycle distribution and apoptosis were analyzed by Flow cytometry. WB was performed to detect the proteinexpressionsofCDK1,CDK2andCaspase-3inHepG2cells. Results: The hypoxic hepatocellular carcinoma model was successfully established. IGF-1R-siRNA-2 showed the most effective interference efficiency and the most significant knockdown of IGF-1R (all P<0.01). The proliferation of HepG2 cells transfected with IGF-1R siRNA-2 was significantly suppressed (P<0.05 or P<0.01), the cell cycle was blocked at G0/G1 phase (P<0.05), and the apoptosis rate was increased up to (25.3±1.3)% P<0.01). In the meanwhile, the expressions of CDK1 and CDK2 were decreased and the expression of Caspase-3 was increased in hypoxic HepG2 cells after IGF-1R knockdown (P<0.05). Conclusion: Interfering IGF-1R by siRNA inhibits the malignant biological behaviors of hypoxic HepG2 cells via regulating cell cycle and apoptosis-related proteins. IGF-1R may be a potential target for the treatment of HCC.
ABSTRACT
Metastasis is one of the main reasons causing death in cancer patients. It was reported that chemotherapy might induce metastasis. In order to uncover the mechanism of chemotherapy-induced metastasis and find solutions to inhibit treatment-induced metastasis, the relationship between epithelial-mesenchymal transition (EMT) and doxorubicin (DOX) treatment was investigated and a redox-sensitive small interfering RNA (siRNA) delivery system was designed. DOX-related reactive oxygen species (ROS) were found to be responsible for the invasiveness of tumor cells in vitro, causing enhanced EMT and cytoskeleton reconstruction regulated by Ras-related C3 botulinum toxin substrate 1 (RAC1). In order to decrease RAC1, a redox-sensitive glycolipid drug delivery system (chitosan-ss-stearylamine conjugate (CSO-ss-SA)) was designed to carry siRNA, forming a gene delivery system (CSO-ss-SA/siRNA) down-regulating RAC1. CSO-ss-SA/siRNA exhibited an enhanced redox sensitivity compared to nonresponsive complexes in 10 mmol/L glutathione (GSH) and showed a significant safety. CSO-ss-SA/siRNA could effectively transmit siRNA into tumor cells, reducing the expression of RAC1 protein by 38.2% and decreasing the number of tumor-induced invasion cells by 42.5%. When combined with DOX, CSO-ss-SA/siRNA remarkably inhibited the chemotherapy-induced EMT in vivo and enhanced therapeutic efficiency. The present study indicates that RAC1 protein is a key regulator of chemotherapy-induced EMT and CSO-ss-SA/siRNA silencing RAC1 could efficiently decrease the tumor metastasis risk after chemotherapy.
ABSTRACT
Metastasis is one of the main reasons causing death in cancer patients. It was reported that chemotherapy might induce metastasis. In order to uncover the mechanism of chemotherapy-induced metastasis and find solutions to inhibit treatment-induced metastasis, the relationship between epithelial-mesenchymal transition (EMT) and doxorubicin (DOX) treatment was investigated and a redox-sensitive small interfering RNA (siRNA) delivery system was designed. DOX-related reactive oxygen species (ROS) were found to be responsible for the invasiveness of tumor cells in vitro, causing enhanced EMT and cytoskeleton reconstruction regulated by Ras-related C3 botulinum toxin substrate 1 (RAC1). In order to decrease RAC1, a redox-sensitive glycolipid drug delivery system (chitosan-ss-stearylamine conjugate (CSO-ss-SA)) was designed to carry siRNA, forming a gene delivery system (CSO-ss-SA/siRNA) downregulating RAC1. CSO-ss-SA/siRNA exhibited an enhanced redox sensitivity compared to nonresponsive complexes in 10 mmol/L glutathione (GSH) and showed a significant safety. CSO-ss-SA/siRNA could effectively transmit siRNA into tumor cells, reducing the expression of RAC1 protein by 38.2% and decreasing the number of tumor-induced invasion cells by 42.5%. When combined with DOX, CSO-ss-SA/siRNA remarkably inhibited the chemotherapy-induced EMT in vivo and enhanced therapeutic efficiency. The present study indicates that RAC1 protein is a key regulator of chemotherapy-induced EMT and CSO-ss-SA/siRNA silencing RAC1 could efficiently decrease the tumor metastasis risk after chemotherapy.
Subject(s)
Female , Humans , Amines/chemistry , Antineoplastic Agents/adverse effects , Breast Neoplasms/pathology , Chitosan/chemistry , Doxorubicin/adverse effects , Drug Delivery Systems , Epithelial-Mesenchymal Transition/drug effects , MCF-7 Cells , Neoplasm Metastasis/prevention & control , Oxidation-Reduction , RNA, Small Interfering/administration & dosage , Reactive Oxygen Species/metabolism , rac1 GTP-Binding Protein/physiologyABSTRACT
Small interfering RNA(siRNA)drugs,a type of nucleic acid drugs,have attracted much attention be- cause of their strong specificity. The siRNA drugs have a broad prospect of therapeutic applications,especially in the an- ti-tumor treatment. However,because the siRNA drugs are essentially nucleotide-based,they have also the same limita- tions as other nucleic acid drugs in application. They are biologically unstable and susceptible to nuclease in vivo,result- ing in poor bioavailability,which restricts their development. Therefore,many studies have been devoted to the develop- ment of siRNA drug delivery systems. As the most abundant protein in human plasma,serum albumin has been widely studied. Albumin has a long circulatory half-life and is non-immunogenic and easy to combine with various kinds of drugs to accumulate in solid tumors. These characteristics make albumin an effective delivery vehicle for siRNA drugs. The re- search progress in siRNA drugs and serum albumin as siRNA drugs delivery vehicles is reviewed in this paper.
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OBJECTIVE: To investigate the role of TREM-1 in tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) secretion from lipopolysaccharide-induced mice macrophage cell lines RAW264.7. METHODS: Designing and synthesizing small interfering RNA (siRNA) with high intangerference ratio, then constructing pLKO1.1-puro-TREM-1 The mice macrophage cell lines RAW264.7 were divided into four groups: control group (control); lipopolysaccharide group (LPS); empty plasmid group (pLKO1.1) - just transfected with pLKO1.1; interference group (siRNA) - transfected with pLKO1.1-puro-TREM1.24 h after stimulation with LPS, real-time PCR was used to detect the mRNA levels of TREM-1, TNF-α and IL-1β respectively. The concentrations of TNF-α and IL-1β were assayed by ELISA. RESULTS: In siRNA group, the mRNA levels of TREM-1, TNF-α and IL-1β were decreased significantly (P<0.01): moreover, the concentrations of TNF-α and IL-1β were lower than other groups significantly (P<0.01). CONCLUSION: Small interfering RNA may reduce TNF-α, IL-1β secretion in LPS-induced macrophage 264.7 through inhibiting the expression of TREM-1 gene.
ABSTRACT
PURPOSE: KAI1 COOH-terminal interacting tetraspanin (KITENIN) has been found to act as a promoter of metastasis in murine models of colon cancer and squamous cell carcinoma (SCC). The suppression of tumor progression and metastasis of established colon cancer in mice was observed after intravenous delivery of small interfering RNA (siRNA) targeting KITENIN. The purpose of this study was to investigate the efficacy of gene therapy targeting KITENIN in human head and neck SCC. MATERIALS AND METHODS: SNU-1041, a well-established human hypopharyngeal SCC cell line, was used. KITENIN expression in SNU-1041 was measured by Western blot analysis. The cells were prepared, maintained in culture dishes with media, and divided into two groups: the si-KITENIN group and the scrambled group (control). The siRNA targeting KITENIN (si-KITENIN) and scrambled DNA were transfected into the SNU-1041 cells in each group. The effect of gene therapy was compared by in vitro experiments to evaluate invasion, migration, and proliferation. RESULTS: KITENIN was strongly expressed in the SNU-1041 cells, and the number of invaded cells was reduced more in the si-KITENIN group than in the scrambled group (p<0.001). The speed for the narrowing gap, made through adherent cells, was lower in the si-KITENIN group (p<0.001), and the number of viable proliferating cells was reduced in the si-KITENIN group compared to the scrambled group (p<0.001, the third day). KITENIN protein expression was no longer identified in the si-KITENIN group. CONCLUSION: Gene therapy using an anti-KITENIN strategy might be effective for head and neck squamous carcinoma.
Subject(s)
Humans , Carcinoma, Squamous Cell/genetics , Carrier Proteins/antagonists & inhibitors , Cell Line, Tumor , Cell Movement , Cell Proliferation , Genetic Therapy , Head and Neck Neoplasms/genetics , Membrane Proteins/antagonists & inhibitors , RNA, Small InterferingABSTRACT
RNA interference(RNAi)is a process by which introduced small interfering RNA(siRNA)can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1(HSV-1)RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40, respectively. In this study, we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.
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@#Objective To observe the effect of small-interfering RNA(siRNA) targeting c-Met,the receptor of hepatocyte growth factor(HGF),on the proliferation of human lens epithelial cells(LECs).Methods siRNA was transferred into LECs cultured in vitro by HiperFect Transfection Reagent.Real-Time PCR was applied to observe the expression of c-Met mRNA in LECs after gene transfer,and MTT assay was used to detect the proliferation of LECs induced by HGF.Results The expression of c-Met mRNA in LECs was significantly decreased in the experimental group,compared to that in the controls(P<0.01).Proliferation of LECs induced by HGF was inhibited,compared with the single HGF stimulated group(P<0.01).Conclusion The RNA interference targeting c-Met can effectively inhibit the expression of c-Met mRNA,and the proliferation of LECs induced by HGF.
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Objective:To study the effect of RNA interference(RNA)ion ATX-mRNA expression and the invasive petential of human breast cancer cell lines.Methods:Chemically synthesizeddouble stranded RNA(dsRNA)targeting ATX was transfected into human breast cancer cell MCF-7 using SiPORT Lipid.The transfection efficiency was observed under fluorescence confocal microscope.Expression of ATX mRNA and protein was detected by reverse transcription polymerase chain reaction(RT-PCR)and Western blot.Cell penetrate matrigel capacity were determined by in vitro experiment.Results:ATX-siRNA effectively inhibited ATX-mRNA and protein expression(P
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Objective:To investigate the expression of CD28 costimulatory molecule on human lymphocytes inhibited by siRNA.Methods:Three different siRNA (siRNA 1,siRNA 2,siRNA 3) were designed and synthysized and transfected into freshly isolated human lymphocytes with cationic liposome.At 24,48 and 72 h post transfection,the changes of CD28 expression were detected by flow cytometry,and the changes of CD28 mRNA levels were determined by semi quantitative RT PCR.Results:Different siRNA showed different reduction in CD28 expression.At 48 h post transfection,the degrees of reduction with siRNA 1,siRNA 2 and siRNA 3 were 22 10%?1 63%,73 50%?1 02% and 42 90%?0 89% respectively compared with the control ( P
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Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-?B ligand (RANKL) and osteoprotegerin (OPG) in cultured human periodontal ligament cells (HPDL cells) as well as its action mechanism. Methods We first constructed small interferring RNA (siRNA) eukaryotic expression vector targeted transforming growth factor ?Ⅱ receptor (TGF-?RⅡ),and then transfected it into T cells. Then HPDL cells together with T cells transfected with siRNA or not were placed in medium that had been added with lipopolysaccharide (LPS) and baicalin. They were divided into six groups and cultured for 48 hours. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to observe the effect of baicalin on OPG-RANKL expression in HPDL cells. Results The clone sequence correctly identified by RT-PCR was consistent with the designed target sequence. The recombinant vector was constructed successfully and the expression of TGF-?ⅡR of T cells which had been transfected with siRNA1 was inhibited obviously. Ratio of RANKL/OPG in each group differed significantly (P